Project Summary Active membrane transport is a critical process for normal cell metabolism, including the maintenance of ion-gradients, osmotic balance, action potentials and apoptosis. The proposed work will address key questions regarding the mechanisms of nutrient uptake in Escherichia coli, and it will address questions regarding the structure and organization of these proteins in the bacterial outer membrane. In E. coli, rare nutrients are sequestered by specific outer- membrane proteins that derive energy by coupling to the inner-membrane protein TonB. These TonB-dependent transporters include BtuB, which is responsible for vitamin B12 transport, and FhuA, FecA and FepA, which are responsible for the transport of various forms of chelated iron. TonB-dependent transporters, such as BtuB, also acts as receptors for antibacterial proteins called colicins, which are produced by bacteria to eliminate other bacteria. TonB- dependent transporters are abundant in Gram negative bacteria and are critical to the proper functioning of the human microbiome as well as the success of many bacterial pathogens, such as those that result in meningitis, cholera, pertussis and dysentery. Because they are unique to bacteria, these transporters are a rational target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for a number of TonB-dependent transporters; however, the mechanism by which transport takes place is unclear. The proposed work will test models for the molecular mechanisms of transport primarily through the use of site-directed spin labeling and EPR spectroscopy. New approaches have been developed to perform double electron-electron resonance in intact E. coli, and these approaches will be used to determine the conformation of TonB-dependent transporters in E. coli under conditions where transport takes place. In the outer membrane, proteins are sequestered into domains or islands, which are thought to drive the turnover of outer-membrane proteins in bacteria. EPR will be used in E. coli to characterize the protein-protein interactions that drive domain formation and define the supramolecular structure of the outer membrane. Finally, EPR on actively metabolizing E. coli will be used to test models for the import of colicin E3 into the bacterial cell.